Effects of Efflux Pump Inhibitors and Antileishmanial Drug Combinations on Leishmania tropica and Leishmania infantum Isolates

Abstract

Drug resistance, one of the most important public health problems facing humanity, necessitates new strategies and approaches in the development of antileishmanial agents. Although developments regarding efflux pump inhibitors (EPIs) and other candidate agents are promising, the search continues to increase the duration of use and efficacy of existing antileishmanials. The aim of this study was to investigate the antileishmanial effects of three EPIs, namely reserpine, berberine and verapamil. The antileishmanial activities of EPIs, pentostam and miltefosine against Leishmania tropica and Leishmania infantum strains were determined by broth microdilution method. Minimum parasiticidal concentration (MPC) values were determined by inverted microscope and IC50 values were determined by MTT viability assay method. The effects of EPIs with determined antileishmanial activities on miltefosine and pentostam were investigated by checkerboard method. The MPC values of antileishmanial drugs miltefosine and pentostam for L.tropica and L.infantum were determined as 64 and 196 pg/mL at 24 and 48 hours, respectively. The MPC values of EPIs reserpine and berberine were determined as 314 and 64 pg/mL for the same incubation times and for verapamil, they were determined as 80 pg/mL at 24 hours and 40 pg/mL at 48 hours. Among anti-leishmanials, the IC50 values of miltefosine were calculated as 4.91/3.47 and 4.05/2.91 pg/mL for L.tropica and L.infantum at 24 and 48 hours, respectively and that of pentostam were calculated as 34.58/59.86 and 18.48/40.63 pg/mL at the same incubation times, respectively.The IC50 values of EPIs, reserpine, berberine, and verapamil were calculated as 74.05/50.61, 7.27/6.1, and 12.52/4.53 pg/mL for L.tropica at 24/48 hours, respectively and 64.52/51.72, 8.21/8.01, and 11.59/7.69 pg/mL for L.infantum. When miltefosine was combined with reserpine, berberine or verapamil, synergistic interactions were observed at 24 and 48 hours of incubation. When pentostam was combined with reserpine, partial synergy was observed at 24 hours and synergy was observed at 48 hours. When pentostam was combined with berberine or verapamil, synergistic interactions were observed at both incubation conditions. Synergy results were found to be the same in both L.tropica and L.infantum strains. In recent years, the speed of research on the discovery of new antimicrobials has significantly decreased and it has become necessary to investigate new molecules that can affect resistance mechanisms. It is thought that EPIs may be a promising approach that can increase the clinical performance of antileishmanial agents and reduce the level of side effects in the fight against drug resistance.